Bacillus thuringiensis is a Gram-positive bacterium that produces delta-endotoxins known as crystal proteins which are specifically toxic to certain orders and species of insects. Many different strains of B. thuringiensis have been shown to produce insecticidal crystal proteins. Compositions including B. thuringiensis strains which produce insecticidal proteins have been commercially available and used as environmentally acceptable insecticides.
The majority of insecticidal B. thuringiensis strains are active against insect of the order Lepidoptera, i.e., caterpillar insects. Other B. thuringiensis strains are active against insects of the order Diptera, i.e., flies and mosquitoes, or against both lepidopteran and dipteran insects. In recent years, a few B. thuringiensis strains have been reported as producing crystal proteins that are toxic to insects of the order Coleoptera, i.e., beetles, such as corn rootworms. Such currently deployed toxic proteins include Cry3Bb1, a modified Cry3A, eCry3.1Ab, and a binary toxin Cry34Ab1/Cry35Ab1 (requiring two different proteins for toxic activity). These proteins are effective for controlling Diabrotica species that infest corn roots, whether deployed singly, or in various combinations to decrease the likelihood of the development of resistance. Even though these proteins have been successfully deployed as insect control agents in transgenic crop plants, resistance to their effects can develop.
The classification of these crystal proteins was previously based on their target insect types. However, ongoing discovery of crystal proteins with very different amino acid sequences and insecticidal activities necessitated the development of a new classification system. The currently accepted nomenclature groups crystal proteins based on their amino acid sequences only. (Crickmore, N. et al. Microbiol. and Mol. Bio. Rev. (1998) Vol. 62: 807-813; http://www.btnomenclature.info/).
Resistance to a deployed toxin, whether chemistry or protein, is more likely to develop in a number of situations which enhance resistance development. Generally, the development of resistance is directly dependent on the length of time that a toxin is deployed into the environment. Resistance development is also more likely to increase in situations in which the dose of the toxin is insufficient to ensure mortality to the pest consuming a single bite of tissue containing the toxin. Accordingly, it is crucial to deliver a lethal dose of toxin with each bite, otherwise development of resistance to a particular toxin is more likely to occur. Repetitive use of the same toxin within a common geographic region on or in multiple species of plants which are susceptible to the same or similar pests within a common geographic region is more likely to cause rapid development of resistance to the toxin, particularly in climates in which there are multiple generations of a particular target pest within a single growing season. For all the forgoing reasons, dependence on a limited number of toxic proteins or toxic chemistries can result in the development of resistance to these pest control agents.
The western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) is a major corn insect pest throughout the United States Corn Belt. The options available for WCR management are limited due to the insect's propensity to adapt to both chemical pesticides and transgenic corn hybrids expressing B.t. Cry proteins, as well as cultural measures such as crop rotation with soybean. In recent years, WCR at specific geographic areas has developed significant resistance to Cry3Bb, likely due to continuously planting Cry3Bb transgenic corn. It has been shown that WCR selected with Cry3Bb is cross resistant to mCry3Aa (Gassmann et al., 2014; Gassmann et al., 2011). Although there has been no report of resistance issue with Cry34/35Ab1 maize traits, Cry34/35 trait is under increasing selection pressure due to larger planting area, e.g. the commercialization of SmartStax, and the possibility of Cry34/35Ab1 selection on top of Cry3-resistant WCR population. The corn rootworm trait market needs novel insecticidal genes with a new mode of action (MOA) for sustainable corn rootworm control (Narva et al., 2013). Other proteins disclosed in the art that are asserted to exhibit toxic effects to corn rootworms include patatin, TIC100/101 binary toxin, ET33/34 binary toxin, TIC863, ET80/76 binary toxin, ET70, Cry3Bb (U.S. Pat. No. 6,501,009), CrylC variants, Cry3A variants, Cry3, Cry3B, Cry34/35, 5307, Axmil84, Axmi205, AxmiRl, TIC901, TIC1201, TIC407, TIC417, TIC431, TIC807, TIC853, TIC3131, eHIPs (U.S. Patent Application Publication No. 2010/0017914), and ω-Hex atoxin-Hv 1a (U.S. Patent Application Publication US2014-0366227 A1).
Despite the discovery of many selective protein toxins from B. thuringiensis, there remains a critical need to discover new, effective pest control agents that provide economic benefits to farmers especially against Diabrotica virgifera virgifera (Western Corn Rootworm (WCR)), and are environmentally acceptable. Particularly needed are agents targeted to control a wide spectrum of economically important insect pests that effectively control insect populations that are, or could become, resistant to existing insect control agents and those with equal to or increased potency compared to currently deployed insecticidal IRDIG protein toxins.